Brain metastases occur in approximately 15% of metastatic breast cancer patients and confer a dismal prognosis. Brain metastases are thought to increasing, particuarly among metastatic patients with Her-2+ or triple negative tumors. Our goals are to identify genes that functionally contribute to brain metastatic progression and to identify and validate preclinical leads. A murine preclinical model of brain metastasis was developed using a derivative of the MDA-MB-231 breast carcinoma cell line (231-BR cells). The proliferative and apoptotic rates exhibited by this cell line, as well as the presence of a neuro-inflammatory response, closely correlate with data from 16 resected brain metastases of human breast cancer, suggesting that the model is relevant. Additional models of brain metastasis of breast cancer have been estalblished to provide a measure of heterogeneity including the murine 4T1 mammary cell line (4T1-BR5), Her-2 transfected human MCF-7 (ER+) cells (MCF-7-Her-2-BR3), and naturally Her-2+ Jimt-1 and SUM190 cells. In collaboration with Drs. Quentin Smith and Paul Lockman, Texas Tech University, the permeability of experimental 231-BR and 4T1-BR5 metastases was quantified. Published collaborative research showed that experimental brain metastases of breast cancer were heterogeneous in their permeability, both within and between metastases in the same brain. While most metastases were permeable as compared to the normal brain blood-brain barrier, 10% exhibited sufficient permeability to enable a cytotoxic response to a systemic drug. Ongoing experiments in the lab investigate the hypothesis that alterations in tumor or brain microenvironmental gene expression underlie altered permeability of metastases. Permeable and impermeable brain lesions have been identified in frozen sections of mouse brains; using the adjacent section the material was laser-capture microdissected. RNA extracted from these lesions was hybridized to both human (tumor cell genes) and mouse (brain microenvironment genes) microarrays. Multiple differentially expressed genes (between permeable and non-permeable brain metastases) have now been identified and are undergoing validation experiments. All are from the microenvironment, rather than the tumor cells. Few if any are standard components of the blood-brain barrier or the drug efflux pump machinery. Leads are being validated at the protein level in two model sytems. We tested the hypothesis that Her-2 overexpression alters the natural history of breast cells to render them more brain metastatic. Her-2 transfectants of the 231-BR cells produced three fold greater large brain metastases, proportional to MRI detectable metastases in a human brain. The efficacy of the dual EGFR/Her-2 tyrosine kinase inhibitor, lapatinib, was tested on the brain metastatic colonization of a human breast carcinoma cell line.). Lapatinib prevented the brain colonization of 231-BR-Her-2 cells by 53% and inhibited the phospho-Her-2 staining of treated brain metastases in vivo, demonstrating that the drug hit its target. Rational combinations with lapatinib were investigated to improve its preventive efficacy. Pazopanib, a VEGFR, PDGFR and c-kit inhibitor, was investigated. Pazopanib as a single agent signficantly prevented the formation of 231-Her2 brain metastases in mice, and was found to exert B-Raf inhibitory activity. Lapatinib/pazopanib combination experiments are under consideration. Pazopanib was also found to affect the neuroinflammatory response to brain metastases, silencing the PDGFR-b activation of a novel subset of astrocytes. Comparison of matched primary tumors and resected brain metastases revealed that Rad51 and another DNA double strand break repair protein, Bard1, were overexpressed in the brain lesions. We have found that transfection of either gene into 231-BR or 4T1-BR cells increases brain metastsis formation by approximately four fold. No effect on lung metastasis was found. The augmentation of brain metastasis by either Rad51 or Bard1 overexpression was found to be dependent on an oxidative response in the brain microenvironment, likely causing DNA damage. Tempol, an oxygen radical scavenger, abrogated the effects of Bard1 and Rad51 overexpression. Current experiments will determine the effect of Bard1 or Rad51 overexpression on radiation therapy. In preclinical experiments, 17 potential therapeutics have been tested for prevention of 231-BR brain metastatic colonization. Of these, only four have demonstrated partial efficacy, highlighting the prohibitive role of the blood-brain barrier. Where tested, the active drugs prevented brain metastases, but statistically failed to treat (shrink) an established brain metastasis. A notable finding is that temozolomide, used in the treatment of primary brain cancers, completely prevented 231-BR experimental brain metastases in an MGMT-dependent manner. MGMT staining of matched primary breast cancers and resected brain metastases was performed collaboratively. Approximately 60% of brain metastases were low to no-MGMT in expression, The data support a brain metastasis prevention trial with temozolomide.